Myeloperoxidase (MPO) is an enzyme with a recognized prognostic role in coronary artery disease (CAD), which is also emerging as a promising biomarker for cardiac risk stratification. However, the lack of a consensus method for its quantification has hindered its implementation in clinical practice. The aim of our work was to optimize an absolute sensitive assay for active MPO without external standards, to validate the method in the clinical context of CAD patients, and to estimate the enzyme specific activity.
In order to determine the MPO concentration using fluorescence readings, this ELISA assay exploits the activity of the enzyme recognized by specific antibodies. The assay was validated in a small cohort of patients that included: healthy subjects (n=60); patients with acute myocardial infarction (AMI, n=25); patients with stable CAD (SCAD, n=25) and a concomitant chronic obstructive pulmonary disease (COPD). Then, total MPO concentration and specific activity (activity/total MPO) were determined.
The assay showed an intra- and inter-assay coefficient of variation of 5.8% and 10.4%, respectively, with a limit of detection (LoD) of 0.074 μU. Both AMI and SCAD patients had higher active and total MPO than controls (p<0.0001 and p<0.01, respectively). The specific activity of MPO was higher in SCAD patients compared to both controls and AMI (p<0.0001).
The study presents a robust and sensitive method for assaying MPO activity in biological fluids with low variability. Moreover, the determination of the specific activity could provide novel insight into the role of MPO in cardiovascular diseases (CVDs).
Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: This study was supported by “Fondo di Ateneo per la Ricerca” (FAR) grant number: 2018-FAR.L-CC_006.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.
1. Nussbaum C, Klinke A, Adam M, Baldus S, Sperandio M. Myeloperoxidase: a leukocyte-derived protagonist of inflammation and cardiovascular disease. Antioxid Redox Signal 2013;18:692–713.10.1089/ars.2012.4783Search in Google Scholar PubMed
2. Strzepa A, Pritchard KA, Dittel BN. Myeloperoxidase: a new player in autoimmunity. Cell Immunol 2017;317:1–8.10.1016/j.cellimm.2017.05.002Search in Google Scholar PubMed PubMed Central
3. Teng N, Maghzal GJ, Talib J, Rashid I, Lau AK, Stocker R. The roles of myeloperoxidase in coronary artery disease and its potential implication in plaque rupture. Redox Rep 2017;22:51–73.10.1080/13510002.2016.1256119Search in Google Scholar PubMed PubMed Central
4. Hansson M, Olsson I, Nauseef WM. Biosynthesis, processing, and sorting of human myeloperoxidase. Arch Biochem Biophys 2006;445:214–24.10.1016/j.abb.2005.08.009Search in Google Scholar PubMed
5. Valacchi G, Virgili F, Cervellati C, Pecorelli A. OxInflammation: from subclinical condition to pathological biomarker. Front Physiol 2018;9:858.10.3389/fphys.2018.00858Search in Google Scholar PubMed PubMed Central
6. Schindhelm RK, Van Der Zwan LP, Teerlink T, Scheffer PG. Myeloperoxidase: a useful biomarker for cardiovascular disease risk stratification? Clin Chem 2009;55:1462–70.10.1373/clinchem.2009.126029Search in Google Scholar PubMed
7. Zhang R, Brennan ML, Fu X, Aviles RJ, Pearce GL, Penn MS, et al. Association between myeloperoxidase levels and risk of coronary artery disease. J Am Med Assoc 2001;286:2136–42.10.1001/jama.286.17.2136Search in Google Scholar PubMed
8. Vita JA, Brennan ML, Gokce N, Mann SA, Goormastic M, Shishehbor MH, et al. Serum myeloperoxidase levels independently predict endothelial dysfunction in humans. Circulation 2004;110:1134–9.10.1161/01.CIR.0000140262.20831.8FSearch in Google Scholar PubMed PubMed Central
9. Daugherty A, Dunn JL, Rateri DL, Heinecke JW. Myeloperoxidase, a catalyst for lipoprotein oxidation, is expressed in human atherosclerotic lesions. J Clin Invest 1994;94:437–44.10.1172/JCI117342Search in Google Scholar PubMed PubMed Central
10. Samsamshariat SZ, Basati G, Movahedian A, Pourfarzam M, Sarrafzadegan N. Elevated plasma myeloperoxidase levels in relation to circulating inflammatory markers in coronary artery disease. Biomark Med 2011;5:377–85.10.2217/bmm.11.28Search in Google Scholar PubMed
11. Cervellati C, Valacchi G, Tisato V, Zuliani G, Marsillach J. Evaluating the link between Paraoxonase-1 levels and Alzheimer’s disease development. Minerva Med 2019;110:238–50.10.23736/S0026-4806.18.05875-5Search in Google Scholar PubMed PubMed Central
12. Franck T, Kohnen S, Boudjeltia KZ, Van Antwerpen P, Bosseloir A, Niesten A, et al. A new easy method for specific measurement of active myeloperoxidase in human biological fluids and tissue extracts. Talanta 2009;80:723–9.10.1016/j.talanta.2009.07.052Search in Google Scholar PubMed
13. Franck T, Minguet G, Delporte C, Derochette S, Zouaoui Boudjeltia K, Van Antwerpen P, et al. An immunological method to combine the measurement of active and total myeloperoxidase on the same biological fluid, and its application in finding inhibitors which interact directly with the enzyme. Free Radic Res 2015;49:790–9.10.3109/10715762.2015.1027197Search in Google Scholar PubMed
14. Gach O, Brogneaux C, Franck T, Serteyn D, Legrand V, Pierard LA, et al. Active and total myeloperoxidase in coronary artery disease and relation to clinical instability. Acta Cardiol 2015;70:522–7.10.1080/AC.70.5.3110512Search in Google Scholar
15. Gelderman MP, Lefkowitz DL, Lefkowitz SS, Bollen A, Moguilevsky N. Exposure of macrophages to an enzymatically inactive macrophage mannose receptor ligand augments killing of Candida albicans. Proc Soc Exp Biol Med 1998;217:81–8.10.3181/00379727-217-44208Search in Google Scholar PubMed
16. Trentini A, Manfrinato MC, Castellazzi M, Tamborino C, Roversi G, Volta CA, et al. TIMP-1 resistant matrix metalloproteinase-9 is the predominant serum active isoform associated with MRI activity in patients with multiple sclerosis. Mult Scler 2015;21:1121–30.10.1177/1352458514560925Search in Google Scholar PubMed
17. Trentini A, Castellazzi M, Cervellati C, Manfrinato MC, Tamborino C, Hanau S, et al. Interplay between matrix metalloproteinase-9, matrix metalloproteinase-2, and interleukins in multiple sclerosis patients. Dis Markers 2016;2016:3672353.10.1155/2016/3672353Search in Google Scholar PubMed PubMed Central
18. Chapman AL, Mocatta TJ, Shiva S, Seidel A, Chen B, Khalilova I, et al. Ceruloplasmin is an endogenous inhibitor of myeloperoxidase. J Biol Chem 2013;288:6465–77.10.1074/jbc.M112.418970Search in Google Scholar PubMed PubMed Central
19. Ansani L, Marchesini J, Pestelli G, Luisi GA, Scillitani G, Longo G, et al. F13A1 gene variant (V34l) and residual circulating FXIIIA levels predict short-and long-term mortality in acute myocardial infarction after coronary angioplasty. Int J Mol Sci 2018;19:1–16.10.3390/ijms19092766Search in Google Scholar PubMed PubMed Central
20. Campo G, Pavasini R, Gallo F, Tonet E, Cimaglia P, Del Franco A, et al. Biological effects of ticagrelor over clopidogrel in patients with stable coronary artery disease and chronic obstructive pulmonary disease. Thromb Haemost 2017;117:1–10.10.1160/TH16-12-0973Search in Google Scholar PubMed PubMed Central
21. Shih J, Datwyler SA, Hsu SC, Matias MS, Pacenti DP, Lueders C, et al. Effect of collection tube type and preanalytical handling on myeloperoxidase concentrations. Clin Chem 2008;54:1076–9.10.1373/clinchem.2007.101568Search in Google Scholar PubMed
22. Naz S, Ghafoor F, Iqbal IA. Effect of collection tube type and freeze–thaw cycles on myeloperoxidase concentrations in blood samples of acute coronary syndrome patients. Ann Clin Biochem 2017;54:348–54.10.1177/0004563216662074Search in Google Scholar PubMed
23. Pulli B, Ali M, Forghani R, Schob S, Hsieh KL, Wojtkiewicz G, et al. Measuring myeloperoxidase activity in biological samples. PLoS One 2013;8:1–10.10.1371/journal.pone.0067976Search in Google Scholar PubMed PubMed Central
24. Vlasova II, Arnhold J, Osipov AN, Panasenko OM. pH-dependent regulation of myeloperoxidase activity. Biochemistry (Mosc) 2006;71:667–77.10.1134/S0006297906060113Search in Google Scholar PubMed
25. Mocatta TJ, Pilbrow AP, Cameron VA, Senthilmohan R, Frampton CM, Richards AM, et al. Plasma concentrations of myeloperoxidase predict mortality after myocardial infarction. J Am Coll Cardiol 2007;49:1993–2000.10.1016/j.jacc.2007.02.040Search in Google Scholar PubMed
26. Dullaart RP, Tietge UJ, Kwakernaak AJ, Dikkeschei BD, Perton F, Tio RA. Alterations in plasma lecithin: cholesterol acyltransferase and myeloperoxidase in acute myocardial infarction: implications for cardiac outcome. Atherosclerosis 2014;234:185–92.10.1016/j.atherosclerosis.2014.02.026Search in Google Scholar PubMed
27. Tretjakovs P, Jurka A, Bormane I, Mikelsone I, Elksne K, Krievina G, et al. Circulating adhesion molecules, matrix metalloproteinase-9, plasminogen activator inhibitor-1, and myeloperoxidase in coronary artery disease patients with stable and unstable angina. Clin Chim Acta 2012;413:25–9.10.1016/j.cca.2011.10.009Search in Google Scholar PubMed
28. Pawlus J, Hotub M, Kozuch M, Dabrowska M, Dobrzyck S. Serum myeloperoxidase levels and platelet activation parameters as diagnostic and prognostic markers in the course of coronary disease. Int J Lab Hematol 2010;32:320–8.10.1111/j.1751-553X.2009.01203.xSearch in Google Scholar PubMed
29. Ndrepepa G, Braun S, Mehilli J, Von Beckerath N, Schömig A, Kastrati A. Myeloperoxidase level in patients with stable coronary artery disease and acute coronary syndromes. Eur J Clin Invest 2008;38:90–6.10.1111/j.1365-2362.2007.01908.xSearch in Google Scholar PubMed
30. Gach O, Magne J, Franck T, Derochette S, Deby G, Serteyn D, et al. Clinical significance of active myeloperoxidase in carotid atherosclerotic plaques. Int J Cardiol 2011;152:149–51.10.1016/j.ijcard.2011.07.068Search in Google Scholar PubMed
31. Bradley PP, Christensen RD, Rothstein G. Cellular and extracellular myeloperoxidase in pyogenic inflammation. Blood 1982;60:618–22.10.1182/blood.V60.3.618.618Search in Google Scholar
32. King CC, Jefferson MM, Thomas EL. Secretion and inactivation of myeloperoxidase by isolated neutrophils. J Leukoc Biol 1997;61:293–302.10.1002/jlb.61.3.293Search in Google Scholar PubMed
33. Deepa M, Pasupathi P, Sankar KB, Rani P, Kumar SP. Free radicals and antioxidant status in acute myocardial infarction patients with and without diabetes mellitus. Bangladesh Med Res Counc Bull 2009;35:95–100.10.3329/bmrcb.v35i3.2999Search in Google Scholar PubMed
34. Grammer TB, Kleber ME, Silbernagel G, Pilz S, Scharnagl H, Lerchbaum E, et al. Copper, ceruloplasmin, and long-term cardiovascular and total mortality (the Ludwigshafen Risk and Cardiovascular Health Study). Free Radic Res 2014;48: 706–15.10.3109/10715762.2014.901510Search in Google Scholar PubMed
35. Aaron SD, Vandemheen KL, Ramsay T, Zhang C, Avnur Z, Nikolcheva T, et al. Multi analyte profiling and variability of inflammatory markers in blood and induced sputum in patients with stable COPD. Respir Res 2010;11:1–12.10.1186/1465-9921-11-41Search in Google Scholar PubMed PubMed Central
36. King PT. Inflammation in chronic obstructive pulmonary disease and its role in cardiovascular disease and lung cancer. Clin Transl Med 2015;4:68.10.1186/s40169-015-0068-zSearch in Google Scholar PubMed PubMed Central
The online version of this article offers supplementary material (https://doi.org/10.1515/cclm-2019-0817).
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